Faculty Bibliography

BACKGROUND: Gene therapy for cancer holds enormous therapeutic promise, but its clinical application has been limited by the inability to achieve targeted, high-level transgene expression with limited systemic toxicity. The authors have developed a novel method for delivering genes to microvascular free flaps (commonly used during reconstructive surgery) to avoid these problems. METHODS: During the finite period in which a free flap is separated from the host (ex vivo), it can be perfused with extremely high titers of genetic material through the afferent artery, resulting in efficient transduction of the tissue. Before reanastomosis, unincorporated genetic material is flushed from the flap, minimizing systemic toxicity. RESULTS: In a rodent model using an adenoviral vector containing the lacZ reporter gene, high regional expression of beta-galactosidase was achieved in all the different cells in a microvascular free flap. Moreover, no beta-galactosidase staining was observed outside of the transduced flap, and viral sequence was undetectable by polymerase chain reaction analysis in other tissues. Further analysis confirmed that high-level transgene expression was precisely localized to the explanted tissue, with no collateral transduction. CONCLUSIONS: Targeting gene delivery with minimal systemic toxicity is essential for successful gene therapy. This form of 'biological brachytherapy' provides a new opportunity to deliver targeted therapeutic transgenes to patients undergoing reconstructive surgery and allows microvascular free flaps to perform therapeutic and reconstructive functions

BACKGROUND: Although microvascular coupling devices are used routinely and successfully for venous anastomosis, there are few published reports demonstrating their efficacy for performing arterial anastomosis. It has been the senior author's (C.Y.A.) preference to perform arterial anastomosis using the microvascular coupling device when feasible. METHODS: All microsurgical breast reconstructions performed by the senior author at the New York University Medical Center between 1998 and 2004 were retrospectively reviewed. A total of 60 patients underwent microsurgical breast reconstruction, of which 20 were bilateral, for a total of 80 flaps. RESULTS: Of the 80 flaps performed, there were 47 muscle-sparing TRAM and 22 deep inferior epigastric perforator (DIEP) flaps, and 11 were superior gluteal flaps. Arterial coupling was successfully performed in 60 of 69 flaps based on the deep inferior epigastric artery (87%) and 2 of 11 gluteal flaps (18%); arterial coupling was performed successfully 62 of 74 times (83.9%) when the thoracodorsal artery was the recipient vessel and never performed when the internal mammary artery was the recipient vessel. The overall flap success rate was 100%. CONCLUSIONS: In our large series, we were able to perform a coupled arterial anastomosis in nearly 80% of the cases, without the loss of any flaps. With proper vessel selection and sufficient experience using the microvascular coupler, arterial coupling may be performed in an expeditious, safe, and reliable fashion with minimal morbidity. Though not commonly practiced, use of the coupling device for arterial anastomosis can provide significant time savings, especially in bilateral breast reconstructions

BACKGROUND: Long-term survival of a skin graft is dependent on eventual revascularization. The authors' aim in the present study was to determine whether skin graft vascularization occurs by (1) simple reconnection of vessels, (2) ingrowth of recipient vasculature, (3) outgrowth of donor-derived vessels, and/or (4) recruitment of bone marrow-derived endothelial progenitor cells. METHODS: Full-thickness skin grafts (1 x 1 cm) were transferred between wild-type FVB/N mice (n = 20) and transgenic tie2/lacZ mice (n = 20), where lacZ expression is controlled by the endothelial specific tie2 promoter, allowing differentiation of recipient and donor endothelial cells. The contribution of endothelial progenitor cells to skin graft neovascularization was determined using a bone marrow transplant model where tie2/lacZ bone marrow was transplanted into wild-type mice (n = 20). RESULTS: Vascular regression in the graft was observed at the periphery starting on day 3 and moving centrally through day 21, sparing graft vessels in the absolute center of the graft. At the same time, vascular ingrowth occurred from the wound bed to replace the regressing vessels. Furthermore, bone marrow-derived endothelial progenitor cells contributed to these new vessels starting as early as day 7. Surprisingly, the contribution of bone marrow-derived vessels to the overall process was approximately 15 to 20 percent of new endothelial cells. CONCLUSIONS: Replacement of the donor graft vasculature by endothelial and endothelial progenitor cells from the recipient along preexisting channels is the predominant mechanism for skin graft revascularization. This mechanism is likely similar for all nonvascularized free grafts and suggests novel strategies for optimizing the vascularization of tissue constructs engineered in vitro

Fibroblasts represent a highly mechanoresponsive cell type known to play key roles in normal and pathologic processes such as wound healing, joint contracture, and hypertrophic scarring. In this study, we used a novel fibroblast-populated collagen lattice (FPCL) isometric tension model, allowing us to apply graded biaxial loads to dermal fibroblasts in a 3-dimensional matrix. Cell morphology demonstrated dose-dependent transition from round cells lacking stress fibers in nonloaded lattices to a broad, elongated morphology with prominent actin stress fibers in 800-mg-loaded lattices. Using quantitative real-time RT-PCR, a dose dependent induction of both collagen-1 and collagen-3 mRNA up to 2.8- and 3-fold, respectively, as well as a 2.5-fold induction of MMP-1 (collagenase) over unloaded FPCLs was observed. Quantitative expression of the proapoptotic gene Bax was down-regulated over 4-fold in mechanically strained FPCLs. These results suggest that mechanical strain up-regulates matrix remodeling genes and down-regulates normal cellular apoptosis, resulting in more cells, each of which produces more matrix. This 'double burden' may underlie the pathophysiology of hypertrophic scars and other fibrotic processes in vivo

In fetal tissues, both soft and hard tissue healing (in long bones) have been found to be scarless. However, healing of membranous bone in the fetal craniofacial skeleton has not been well documented. Pregnant ewes (gestational age range, 80-95 days) underwent a hysterotomy, and fetal lambs had a full-thickness excision of the entire mandibular symphysis region (10 mm). Nonoperated controls were used for comparison (n = 8). After 10 days and 2 weeks, fetuses showed incomplete regeneration of the anterior mandible by examination, computed tomographic scan, and histology. By 4 weeks postoperatively, the mandibular defect had completely closed, but regenerated bony volume was less than control specimens. At 6 weeks postoperatively, the specimen demonstrated complete bony healing without scar or inflammation. Computed tomographic scan measurements for mandibular shape (length over width) was similar in experimental and control specimens. The data indicate that fetal lamb membranous bone defects heal in a scarless fashion and suggest preprogrammed migration of osteogenic tissue

Cerebrospinal fluid (CSF) drainage catheters can cause a myriad of complications, in large part because they may migrate from their normal location to almost anywhere in the body. We present the unique case of a female patient who had previously undergone bilateral breast augmentation who experienced sudden painless swelling of her right breast 6 weeks after placement of a ventriculoperitoneal shunt. Radiologic examination demonstrated ensnarement of the distal aspect of the shunt around her implant, with subsequent formation of a CSF pseudocyst. Management of this patient included replacement of the shunt, drainage of the CSF pseudocyst, and preservation of the implant

The trafficking of circulating stem and progenitor cells to areas of tissue damage is poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1 or CXCL12) mediates homing of stem cells to bone marrow by binding to CXCR4 on circulating cells. SDF-1 and CXCR4 are expressed in complementary patterns during embryonic organogenesis and guide primordial stem cells to sites of rapid vascular expansion. However, the regulation of SDF-1 and its physiological role in peripheral tissue repair remain incompletely understood. Here we show that SDF-1 gene expression is regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1) in endothelial cells, resulting in selective in vivo expression of SDF-1 in ischemic tissue in direct proportion to reduced oxygen tension. HIF-1-induced SDF-1 expression increases the adhesion, migration and homing of circulating CXCR4-positive progenitor cells to ischemic tissue. Blockade of SDF-1 in ischemic tissue or CXCR4 on circulating cells prevents progenitor cell recruitment to sites of injury. Discrete regions of hypoxia in the bone marrow compartment also show increased SDF-1 expression and progenitor cell tropism. These data show that the recruitment of CXCR4-positive progenitor cells to regenerating tissues is mediated by hypoxic gradients via HIF-1-induced expression of SDF-1

Pulsed electromagnetic fields (PEMF) have been shown to be clinically beneficial, but their mechanism of action remains unclear. The present study examined the impact of PEMF on angiogenesis, a process critical for successful healing of various tissues. PEMF increased the degree of endothelial cell tubulization (sevenfold) and proliferation (threefold) in vitro. Media from PEMF cultures had a similar stimulatory effect, but heat denaturation ablated this activity. In addition, conditioned media was able to induce proliferative and chemotactic changes in both human umbilical vein endothelial cells and fibroblasts, but had no effect on osteoblasts. Angiogenic protein screening demonstrated a fivefold increase in fibroblast growth factor beta-2 (FGF-2), as well as smaller increases in other angiogenic growth factors (angiopoietin-2, thrombopoietin, and epidermal growth factor). Northern blot analysis demonstrated an increase in FGF-2 transcription, and FGF-2 neutralizing antibody inhibited the effects of PEMF. In vivo, PEMF exposure increased angiogenesis more than twofold. We conclude that PEMF augments angiogenesis primarily by stimulating endothelial release of FGF-2, inducing paracrine and autocrine changes in the surrounding tissue. These findings suggest a potential role for PEMF in therapeutic angiogenesis

Gene therapy is a promising modality for the treatment of soft tissue malignancies. Our laboratory has developed a novel technique of gene transfer using microvascular free flaps that addresses many of the current barriers preventing gene therapy from achieving widespread clinical use. Our previous work has demonstrated our ability to transduce free flaps with an adenovirus encoding the reporter gene lacZ. In this current study, we show that microvascular free flaps can be transduced with an adenovirus encoding the angiogenesis inhibitor endostatin with high levels of local flap expression. These transduced free flaps were able to serve as 'biologic pumps' and were able to secrete endostatin into the serum as demonstrated by enzyme-linked immunosorbent assay. This form of 'biologic brachytherapy' could provide a novel approach for the continuous delivery of therapeutic genes to a localized area while avoiding many of the practical obstacles currently limiting gene therapy

The goal of animal wound healing models is to replicate human physiology and predict therapeutic outcomes. There is currently no model of wound healing in rodents that closely parallels human wound healing. Rodents are attractive candidates for wound healing studies because of their availability, low cost, and ease of handling. However, rodent models have been criticized because the major mechanism of wound closure is contraction, whereas in humans reepithelialization and granulation tissue formation are the major mechanisms involved. This article describes a novel model of wound healing in mice utilizing wound splinting that is accurate, reproducible, minimizes wound contraction, and allows wound healing to occur through the processes of granulation and reepithelialization. Our results show that splinted wounds have an increased amount of granulation tissue deposition as compared to controls, but the rate of reepithelialization is not affected. Thus, this model eliminates wound contraction and allows rodents' wounds to heal by epithelialization and granulation tissue formation. Given these analogies to human wound healing, we believe that this technique is a useful model for the study of wound healing mechanisms and for the evaluation of new therapeutic modalities